There is a critical need for fiber-reinforced tissue engineered constructs (TECs) to function in the hostile environments encountered by fibrous load-bearing tissues. The objective of this renewal is to fabricate and grow a fiber-reinforced TEC for the annulus fibrosus (AF) that meets quantitative design criteria set by the native tissue structure-function properties. Our group has taken the approach of first developing a detailed understanding of the native tissue's structure-function relationships by integrating sophisticated and rigorous mechanical testing with mathematical modeling. Fiber-reinforced scaffolds are key components to achieve TEC mechanical design requirements and influence tissue growth. Our group uses aligned electrospun nanofibrous scaffolds to prescribe mechanical properties and tissue architecture to enhance structure-function properties over time in culture. In the following Aims we combine our expertise in tissue mechanics, mathematical modeling, fabrication of aligned nanofibrous scaffolds, and tissue engineering, with standard measures of biochemistry and histology to generate functional load-bearing fiber-reinforced tissue equivalents. Aim 1: Quantify native human AF tissue structure-function under complex loading to establish the TEC design requirements. Aim 2: Create a single layer TEC from an aligned electrospun nanofibrous scaffold seeded with AF cells. Aim 3: Create a planar stacked TEC from pre-seeded nanofibrous scaffolds with alternating fiber orientation in each layer to mimic native AF architecture. This study will achieve a functional tissue engineered AF, with the ultimate goal of fabricating a full structural engineered disc and clinical implementation as a total disc implant. Innovations include our approach to functional tissue engineering, focusing first on benchmark TEC mechanics and using constitutive modeling to set design criteria. Our aligned nanofibrous scaffold and our `layered' approach, mimicking the native AF tissue structure are also innovative. Notably, this design approach for load-bearing fiber-reinforced tissues, focusing on mechanics first, can be extended to other orthopaedic and cardiovascular tissue applications. PUBLIC HEALTH RELEVANCE. There is a critical need for fiber-reinforced tissue engineered constructs (TECs) to function in the hostile environments encountered by fibrous load-bearing tissues. The objective of this renewal is to fabricate and grow a fiber-reinforced TEC for the annulus fibrosus that meets quantitative design criteria set by the native tissue structure-function properties. We combine our expertise in tissue mechanics, mathematical modeling, fabrication of aligned nanofibrous scaffolds, and tissue engineering to generate functional load-bearing fiber- reinforced tissue equivalents. [unreadable] [unreadable] [unreadable]